Monoamine oxidase (MAO) is one of the enzymes responsible for the catabolism of various biogenic amines such as norepinephrine, serotonin, and dopamine. It has been shown in chronically-depressed individuals that the concentration of various biogenic amines is diminished. Consequently, compounds that inhibit or inactivate MAO A, the isozyme that selectively degrades norepinephrine and serotonin, exhibit antidepressant activity. Because of the importance of maintaining a high concentration of dopamine in the brains of those afflicted with Parkinson disease, compounds that inactivate MAO B, the isozyme that degrades dopamine, are used in the treatment of Parkinson's disease. The specific aims for this project period are to elucidate further the catalytic mechanism of MAO, to design new inactivators and to determine their mechanisms of inactivation, to identify residues at the active site of the enzyme, and to develop applications of utilizing MAO in organic solvents. Efforts directed at the catalytic mechanism of MAO will include the study of the reaction of MAO with (aminomethyl)cubane (both enzymatically and nonenzymatically) and use of compounds containing nitrone groups to trap radicals generated in the molecules. A compound containing an epoxide will be prepared to test a radical versus an anionic mechanism. Stopped-flow kinetics experiments are proposed to try to detect radical intermediates during turnover. The reversibility of the MAO-catalyzed reaction will be determined by running the reaction backwards from the imine with reduced MAO in organic solvents. The mechanism of inactivation of MAO by benzyl (1- aminomethyl)cyclopropanecarboxylate and by a 3-aminomethyl-5-aryl-2- furanone will be studied and the structure of the active site adducts formed upon inactivation by each will be elucidated. A proposed inactivation mechanism by 1-phenylcyclopropylamine will be tested by synthesis of chirally monodeuterated analogues and investigation of specific deuterium isotope effects of the reaction. Inactivation of MAO by 5-aryl-3-dimethylaminomethyl-2-oxazolidinone will be investigated by high field NMR spectroscopy of a 13/C-labeled analogue. A series of oxaheptylamines will be synthesized to test the inductive effect hypothesis of MAO inactivation and to determine the distance that an electron-withdrawing group will still exhibit an effect on stabilizing an adduct. Six new potential inactivators of MAO are proposed, one a cubane analogue, three based on heteroatom inductive effects, and two requiring a radical rearrangement. Four radioactively-labeled inactivators of MAO, (aminomethyl)cubane, 1-phenylcyclopropylamine, N-cyclopropyl-N-alpha- methylbenzylamine, and N-2-aminoethyl-4-chlorobenzamide, will be synthesized and used to label the active site. Peptide mapping will be carried out to determine an active-site peptide. A new substrate for MAO which generates a fluorescent product will be developed for studies with whole cells. The use of MAO as a catalyst for organic synthesis in organic solvents will be explored.